Control of the worldwide HIV epidemic will require development of a preventive and therapeutic HIV vaccine. The immune evasion capabilities of HIV have made vaccine development difficult. An effective vaccine will need to elicit neutralizing antibodies as well as an effective CTL response. Toll-like receptor (TLR) ligands are potent activators of dendritic cells and other antigen presenting cells that are central in the development of a successful adaptive immune response. We hypothesize that using synthetic TLR ligands, such as immunostimulatory DNA, in HIV vaccine preparations will improve anti-HIV immune responses. Furthermore, inhibiting TLR-induced factors that down-regulate immune responses, such as IL-10 and indoleamine 2,3-dioxygenase (IDO), should further optimize vaccine responses. We propose to investigate these hypotheses using the following approaches: 1) Development of memory CD8+ T cell responses after TLR ligand-based vaccination with model antigens (ovalbumin and inactivated SIV) will be assessed in mice by following CTL responses, characterizing development of effector and central memory responses, and determining the requirement for CD4+ T cells in the development and maintenance of memory cells. 2) The ability to augment immune responses by inhibiting IDO, a critical enzyme in tryptophan catabolism that down-regulates T cell responses, and the inhibitory cytokine IL-10 will be tested by assessing the effect of inhibitors and knockouts on in vitro and in vivo immune responses after treatment with TLR-based vaccines. 3) The ability to induce immune responses against SIV will be tested in mice that have been depleted of various cell types, including CD4+ T cells and TLR9+ APCs, to mimic SIV infection. 4) The efficaciousness of TLR ligand-based vaccines in human systems will be assessed by using human PBMCs. Augmentation of CD4+ and CD8+ T cell memory responses to CMV in CMV+ donors after incubation with TLR ligands and inactivated CMV will be assessed. Subsequently, the effect of CD4-depletion will be determined on anti-CMV responses and anti-HIV responses by using PBMCs from HIV+/CMV+ donors. The proposed experiments are designed to provide a balanced investigation that explores fundamental immunology and provides the practical aspects of vaccinology that will move us closer to development of an effective HIV vaccine. [unreadable] [unreadable]